Na,K-ATPase is an ubiquitous plasma membrane-derived enzyme which establishes and maintains the electrochemical gradient of Na and K ions across the membranes of most animal cells. This enzyme is also the target enzyme for a class of drugs known as cardiac glycosides, which are used to treat congestive heart failure. Despite the widespread use of these drugs, little is known about the nature of the binding site on the enzyme. The long-term goal of this research is the detailed analysis of the cardiac glycoside binding site on Na,K-ATPase, as well as the characterization of the membrane organization of the enzyme's alpha subunit. The aim of identifying intracellular versus extracellular regions will aid in the generation of a model of the entire enzyme, as well as of the cardiac glycoside binding site, which is known to be extracellular. The approach will utilize a combination of biochemical, immunological and molecular biological methodologies. All of the aims of the proposal entail the use of in vitro mutagenesis to introduce amino acid substitutions in the a subunit of the Na,K-ATPase. These mutants will be expressed in a culture system that has been devised to facilitate the selection of only those mutants that are still biologically active. One aspect of the proposal entails the use of site-directed and random mutagenesis to identify specific amino acids of the Na,K-ATPase alpha subunit that are involved in cardiac glycoside binding. Mutants will be made and transfected into HeLa cells. Cells will be selected in ouabain and if a mutation has been made that interferes with binding, then resistant cells will be generated. In the. case of random mutagenesis, the polymerase chain reaction will be used to identify the amino acid change that mediates the resistant phenotype. In addition, existing ouabain-resistant site-directed mutants, as well as those identified in this project, will be characterized in detail regarding the inhibition potency of several different glycoside analogues and aglycones in an effort to provide insight into what specific regions of Na,K-ATPase interact with particular structural features of the inhibitor molecule. Finally, mutagenesis will be used to introduce an 8 amino acid "flag" sequence into various regions of the alpha subunit. The flag sequence is not only an epitope for a monoclonal antibody, but is a unique specific protease site as well. The flagged alpha subunit will be expressed in HeLa cells. Following the selection of biologically active mutants, intact or permeabilized cells will be incubated with either the monoclonal antibody or the protease. Immunofluorescence or protein immunoblots will be used to determine it the protein reagent was able to interact with its target sequence in intact versus permeabilized cells. Mutagenesis will also be used to study the spatial organization of the extracellular domains of the alpha subunit.